Project Summary In vitro development of highly organized and vascularized three-dimensional (3D) tissue constructs is of great importance in tissue engineering, since native muscle tissues exhibit highly organized 3D complex architectures composed of an extracellular matrix (ECM), different cell types, and chemical and physical signaling cues. Bioprinting has emerged as a new technology to develop highly complex, 3D structures; however, there are many remaining challenges, such as the necessity for precise positioning/switching of different cell-types and materials to create multi-cellular 3D structures with various sizes, and creating patterns that resemble the physical properties of in vivo environments. To address these challenges, we plan to develop an embedded multi-material bioprinting (EMB) technology that employs a self-healing supporting hydrogel and a programmable microfluidic device. The multi-material bioprinting (MB) system can be developed by integration of a direct-write 3D bioprinting system with a high precision, programmable microfluidic printhead, which can easily and quickly switch between different materials, reagents and cells. The multi-axial extrusion systems are able to create multi-scale microfibers for muscle bundles and perfusable blood vessel networks to mimic the mechanical properties and architecture of their spatially organized natural counterparts. While it is difficult to precisely control the materials? position in Z directions to create freestanding hydrogel architectures, we will improve the high print fidelity of the MB system by combining an embedded 3D bioprinting technology by using a self-healing supporting hydrogel. In addition, the supporting hydrogel will be able to achieve fast deposition of the desired pre-polymer solution in X-Y-Z directions without additional gelation processing. By combining this embedded printing strategy with the microfluidic device incorporated MB technology, it will allow us to print multi-component/multi-cellular tissue constructs with biologically relevant architectures and characteristics that are difficult or impossible to bioprint at present. Furthermore, the use of a cell-laden bioink, which mimics the mechanical and biological properties of muscle tissue, can act as a platform to promote differentiation and maturation of muscle precursors, as well as improved contractile activity. It is envisioned that the successful development of this project will have a significant impact on the ability to heal muscle trauma as well as to advance the field of muscle tissue engineering. Furthermore, this process can be readily applied to other areas of regenerative medicine to generate new organs.